Summary Brassica napus is highly susceptible towards Verticillium longisporum ( Vl43 ) with no effective genetic resistance. It is believed that the fungus reprogrammes plant physiological processes by up‐regulation of so‐called susceptibility factors to establish a compatible interaction. By transcriptome analysis, we identified genes, which were activated/up‐regulated in rapeseed after Vl43 infection. To test whether one of these genes is functionally involved in the infection process and loss of function would lead to decreased susceptibility, we firstly challenged KO lines of corresponding Arabidopsis orthologs with Vl43 and compared them with wild‐type plants. Here, we report that the KO of AtCRT1a results in drastically reduced susceptibility of plants to Vl43 . To prove crt1a mutation also decreases susceptibility in B. napus , we identified 10 mutations in a TILLING population. Three T3 mutants displayed increased resistance as compared to the wild type. To validate the results, we generated CRISPR/Cas‐induced BnCRT1a mutants, challenged T2 plants with Vl43 and observed an overall reduced susceptibility in 3 out of 4 independent lines. Genotyping by allele‐specific sequencing suggests a major effect of mutations in the CRT1a A‐genome copy, while the C‐genome copy appears to have no significant impact on plant susceptibility when challenged with Vl43 . As revealed by transcript analysis, the loss of function of CRT1a results in activation of the ethylene signalling pathway, which may contribute to reduced susceptibility. Furthermore, this study demonstrates a novel strategy with great potential to improve plant disease resistance.

Fusarium graminearum, the most important causal agent of head blight of cereals, produces mycotoxins like the trichothecene deoxynivalenol (DON). This fungus was resistant towards the fungicide trifloxystrobin and externally applied DON whereas it was sensitive towards the demethylation inhibitors epoxiconazole and tebuconazole. Forty-six expressed sequence tags (ESTs) were screened for induction after exposure of mycelium to sub-lethal concentrations of trifloxystrobin, epoxiconazole, tebuconazole, or DON. Four putative transporter genes were up-regulated more than 3-fold after treatment with trifloxystrobin, a fungicide derived from a natural toxin produced by fungi of the genus Strobilurus. Interestingly, the same four genes were also transcriptionally induced by externally applied DON. In contrast, no significant transcriptional induction of the four candidate genes was observed after exposure of the fungus to epoxiconzole and tebuconazole. Two of the up-regulated genes belong to the MFS-transporter family (FGSG_01584; FGSG_07802), one is a putative ABC-transporter (FGSG_17395), the fourth is a probable polypeptide-transporter (FGSG_07227).

The cyst nematode Heterodera schachtii belongs to the most important biotic stress factors in sugar beet cultivation. Nematodes can be controlled by crop rotation, by fumigation with nematicides or by growing resistant crops. However, nematicides are no longer admitted to be used because of their toxic environmental impacts, the crop rotation system is often agronomically impracticable and the genetic resistance is so far not available in sugar beet germplasms. Thus, alternative strategies for nematode control have been applied in the past years. For instance, genetic resistance from wild beet species were identified and transferred into the elite breeding materials. Today several nematode tolerant varieties of sugar beet are commercially available. In addition, different approaches based on genetic engineering have been developed to improve plant nematode resistance. Nevertheless, an effective control of nematodes in sugar beet cultivation still remains a great challenge.

Nematode (Heterodera schachtii) resistance in sugar beet (Beta vulgaris) is controlled by a single dominant resistance gene, Hs1(pro-1). BvGLP-1 was cloned from resistant sugar beet. The BvGLP-1 messenger (m)RNA is highly upregulated in the resistant plants after nematode infection, suggesting its role in the Hs1(pro-1) mediated resistance. BvGLP-1 exhibits sequence homology to a set of plant germin-like proteins (GLP), from which several have proved to be functional in plant basal or defense resistance against fungal pathogens. To test whether BvGLP-1 is also involved in the plant-fungus interaction, we transferred BvGLP-1 into Arabidopsis and challenged the transgenic plants with the pathogenic fungi Verticillium longisporum and Rhizoctonia solani as well as with the beneficial endophytic fungus Piriformospora indica. The expression of BvGLP-1 in Arabidopsis elevated the H(2)O(2) content and conferred significant resistance to V. longisporum and R. solani but did not affect the beneficial interaction with P. indica in seedlings. Microscopic observations revealed a dramatic reduction in the amount of hyphae of the pathogenic fungi on the root surface as well as of fungal mycelium developed inside the roots of transgenic Arabidopsis compared with wild-type plants. Molecular analysis demonstrated that the BvGLP-1 expression in Arabidopsis constitutively activates the expression of a subset of plant defense-related proteins such as PR-1 to PR-4 and PDF1.2 but not PDF2.1 and PDF2.3. In contrast, the PDF2.1 mRNA level was downregulated. These data suggest an important role of BvGLP-1 in establishment of plant defense responses, which follow specific signaling routes that diverge from those induced by the beneficial fungus.

Crops are attacked by parasitic pests, including insects and nematodes causing considerable economic losses worldwide. The global yield loss of crops due to herbivorous insects varies between 5% and 30% depending on crop species, while the economic losses due to plant parasitic nematodes are about US $125 billion annually. Today, the control of plant parasites mainly depends on relatively few chemicals that pose serious concerns of risks and hazards for humans, animals and the environment and also increase the costs of growing crops. Use of natural resistance mechanisms offers a promising alternative for parasite control. A set of resistance genes has been identified. Advanced understandings of natural resistance mechanisms in molecular details broaden the horizon of crop resistance breeding programs. Because the resistance resource is limited in many crop species and gene-for-gene reliant resistance is easily overcome by new virulent pathotypes, new genetic variability is needed. Therefore, engineered resistance is becoming an essential part of a sustainable parasite control as it offers a parasite management with benefits to the producer, the consumer and the environment. For engineering resistance , several approaches are under discussion and application. This review focuses on the strategy for engineering parasite resistance in crops by genetic modification.

The majority of known plant resistance genes encode proteins with conserved nucleotide-binding sites and leucine-rich repeats (NBS-LRR). Degenerate primers based on conserved NBS-LRR motifs were used to amplify analogues of resistance genes from the dicot sugar beet. Along with a cDNA library screen, the PCR screen identified 27 genomic and 12 expressed NBS-LRR RGAs (nlRGAs) sugar beet clones. The clones were classified into three subfamilies based on nucleotide sequence identity. Sequence analyses suggested that point mutations, such as nucleotide substitutions and insertion/deletions, are probably the primary source of diversity of sugar beet nlRGAs. A phylogenetic analysis revealed an ancestral relationship among sugar beet nlRGAs and resistance genes from various angiosperm species. One group appeared to share the same common ancestor as Prf, Rx, RPP8, and Mi, whereas the second group originated from the ancestral gene from which 12C1, Xa1, and Cre3 arose. The predicted protein products of the nlRGAs isolated in this study are all members of the non-TIR-type resistance gene subfamily and share strong sequence and structural similarities with non-TIR-type resistance proteins. No representatives of the TIR-type RGAs were detected either by PCR amplification using TIR type-specific primers or by in silico screening of more than 16,000 sugar beet ESTs. These findings suggest that TIR type of RGAs is absent from the sugar beet genome. The possible evolutionary loss of TIR type RGAs in the sugar beet is discussed.

The Hs1 pro−1 gene confers resistance to the beet cyst nematode Heterodera schachtii in sugar beet ( Beta vulgaris L.) on the basis of a gene-for-gene relationship. RNA-gel blot analysis revealed that the transcript of Hs1 pro−1 was present in uninfected roots of resistant beet at low levels but increased by about fourfold one day after nematode infection. Treatments of plants with external stimuli including salicylic acid, jasmonic acid, gibberellic acid and abscisic acid as well as wounding or salt stress did not result in changes in the gene transcription, indicating de novo transcription of Hs1 pro−1 upon nematode infection specifically. To study transcriptional regulation of Hs1 pro−1 expression at the cellular level, a 3082 bp genomic fragment representing the Hs1 pro−1 promoter, isolated from the YAC-DNA housing the Hs1 pro−1 gene, was fused to the β-glucuronidase reporter gene (1832prm1::GUS) and transformed into susceptible beet roots and Arabidopsis plants, respectively. Fluorometric and histochemical GUS assays on transgenic beet roots and Arabidopsis plants carrying the 1832prm1::GUS construct demonstrated that the Hs1 pro1 promoter is functional in both species and drives a nematode responsive and feeding site-specific GUS-expression. GUS activity was detected as early as at initiation of the nematode feeding sites and GUS staining was restricted to the nematode feeding sites. To delineate the regulatory domains of the Hs1 pro−1 promoter, fusion genes with various 5′ deletions of the Hs1 pro−1 promoter and the GUS gene were constructed and analysed in transgenic beet roots as well. Cis elements responsible for feeding site-specific gene expression reside between –355 and +247 from the transcriptional initiation site of Hs1 pro−1 whereas an enhancer region necessary for higher gene expression is located between −1199 and −705 of the promoter. The Hs1 pro−1 promoter drives a nematode feeding site-specific GUS expression in both sugar beet and Arabidopsis suggesting a conserved mechanism of regulation of Hs1 pro−1 expression in these two species.

Sporamin, a sweet potato tuberous storage protein, is a Kunitz-type trypsin inhibitor. Its capability of conferring insect-resistance on transgenic tobacco and cauliflower has been confirmed. To test its potential as an anti-feedant for the beet cyst nematode (Heterodera schachtii Schm.), the sporamin gene SpTI-1 was introduced into sugar beet (Beta vulgaris L.) by Agrobacterium rhizogenes-mediated transformation. Twelve different hairy root clones expressing sporamin were selected for studying nematode development. Of these, 8 hairy root clones were found to show significant efficiency in inhibiting the growth and development of the female nematodes whereas 4 root clones did not show any inhibitory effects even though the SpTI-1 gene was regularly expressed in all of the tested hairy roots as revealed by northern and western analyses. Inhibition of nematode development correlated with trypsin inhibitor activity but not with the amount of sporamin expressed in hairy roots. These data demonstrate that the trypsin inhibitor activity is the critical factor for inhibiting growth and development of cyst nematodes in sugar beet hairy roots expressing the sporamin gene. Hence, the sweet potato sporamin can be used as a new and effective anti-feedant for controlling cyst nematodes offering an alternative strategy for establishing nematode resistance in crops.